Abstract
Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from V(H), D, and J(H) gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a J(H)-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to J(H)s to form a DJ(H)-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the V(H) and D/J(H) domains, over 100 CBEs across the V(H) domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and V(H) CBEs. IGCR1 CBEs segregate D/J(H) and V(H) domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJ(H)-RC-bound RAG to scan the V(H) domain and perform V(H)-to-DJ(H) rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-J(H) to V(H)-to-DJ(H) recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.